Abstract

Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic “reader” proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction.

SIGNIFICANCE STATEMENT Proteins involved in the “readout” of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and BET inhibitors are currently being studied in several clinical trials. However, their role in addiction-related phenomena remains unknown. In the current studies, we revealed that BRD4 is elevated in the nucleus accumbens and recruited to promoter regions of addiction-related genes following repeated cocaine administration, and that inhibition of BRD4 attenuates transcriptional and behavioral responses to cocaine. Together, these studies reveal that BET inhibitors may have therapeutic utility in the treatment of cocaine addiction.

The bromodomain and extraterminal domain (BET) family of proteins (BRD2, BRD3, BRD4, and BRDT) contain two bromodomains, which bind acetylated histone tails and are involved in transcriptional coactivation and elongation (Dhalluin et al., 1999; Winston and Allis, 1999; Owen et al., 2000; Patel et al., 2013). In particular, BET proteins have high affinity for clustered polyacetylated histone lysine sites but also bind to monoacetylated lysines with modest affinity (Dey et al., 2003; Morinière et al., 2009). Genome-wide analysis studies have revealed that BET-bound nucleosomes are enriched in histone post-translational modifications that regulate actively transcribed euchromatin, and BRD4-bound sites are highly correlated with increased gene expression (LeRoy et al., 2012) and play a role in super-enhancer function (Zhang et al., 2012; Brown et al., 2014). With the development of selective, small-molecule inhibitors of BET bromodomains (Filippakopoulos et al., 2010; Chung et al., 2011), there has been increasing interest in their therapeutic utility (Delmore et al., 2011; Zuber et al., 2011; Belkina and Denis, 2012; Barrett et al., 2014), and inhibitors of these proteins are currently being tested in clinical trials for treatment of several diseases (www.Clinical-trials.gov; IDs NCT01713582, NCT01949883, NCT02157636, NCT01987362, and NCT01587703). However, it is unclear whether BET proteins regulate molecular and behavioral aspects of addiction. Given that histone acetylation mechanisms regulate cocaine-induced neuroadaptations and behaviors (LaPlant and Nestler, 2011), we hypothesized that BET proteins may also play a vital role in addiction-related phenomena. Using behavioral, pharmacological, and molecular techniques, we show that BET proteins are upregulated and recruited to promoter region of Bdnf in the NAc following repeated cocaine administration and that inhibition of these proteins attenuates transcriptional and behavioral responses to cocaine. Together, these studies indicate that the displacement of BET proteins from chromatin may have therapeutic efficacy in addiction-related behaviors.

Materials and Methods

Animals

Male C57BL/6 mice (8–10 weeks old) and Sprague Dawley rats (initial weight ∼300–325 g, Charles River Laboratories) were housed 2–4 animals per cage under a regular 12 h/12 h light/dark cycle and had ad libitum access to food and water. Rats that received cannula or catheter surgeries were single-housed following surgery to prevent cage-mates from tampering with catheter/cannula implants. Mice and rats were housed in a humidity and temperature-controlled, Association for Assessment and Accreditation of Laboratory Animal Care-accredited, animal facility at the University of Miami Miller School of Medicine. All experiments were approved by the Institutional Animal Care and Use Committee before experimentation and conducted according to specifications of the National Institutes of Health as outlined in the Guide for the Care and Use of Laboratory Animals.

Drug treatments

Cocaine HCl (National Institute on Drug Abuse) was dissolved in 0.9% sterile saline. Mice and rats received one injection (intraperitoneally) of saline or cocaine (10 mg/kg for rats and 20 mg/kg for mice) per day for 10 d, and were killed at multiple time points following the last injection. The BET inhibitor, JQ1 or iBET-151 (Tocris Bioscience), was dissolved in 10% DMSO and 10% 2-hydroxypropyl-β-cyclodextrin in sterile PBS; 10–50 mg/kg was given in a volume of 0.08–0.12 ml before the cocaine conditioning session. Vehicle was delivered at the same volume as the JQ1/iBET-151 solution. For intracranial injections, JQ1 (10 μm) was dissolved in aCSF (Tocris Bioscience).

Cell culture

Neuro-2a (N2a) and HEK293 cell lines (ATCC) were maintained in DMEM supplemented with 10% FBS and 1% primocin. In a 6 well plate, ∼400,000 cells were plated and treated 24 h later with DMSO (vehicle) or JQ1 (100 nm or 1 μm) for 48 h. In HEK293 cells, gene silencing was achieved by reverse transfection of scrambled control, BRD2-, BRD3-, or BRD4-siRNA (20 nm for 48 h) in lipofectamine RNAiMax (Invitrogen) according to the manufacturer's instructions. These previously validated BET siRNAs (Pastori et al., 2015) were purchased from Ambion (siBRD4-s23901, siBRD3-s15545, and siBRD2-s12070), and the control siRNA was purchased from QIAGEN (SI03650318). Cells were harvested for RNA and/or protein analysis as described below.

Surgeries

Intravenous catheter implantation.

Rats were anesthetized with an intraperitoneal injection of a ketamine/xylazine mixture (60–80 mg/kg/10 mg/kg). In cocaine self-administration studies, rats were implanted with chronic indwelling intravenous catheters where sterile Silastic tubing was inserted into the jugular vein and the other end of the tubing passed subcutaneously over the shoulder blades to a cannula mounted on the back. Intravenous catheters were flushed once a day with the antibiotic cefazolin (10 mg, i.v.) and heparin (10 U, i.v.). After 5–7 d of recovery, rats received self-administration training.

Guide cannula implantation.

Anesthetized rats were stereotaxically implanted with bilateral guide cannulae (22 gauge, Plastics One) aimed 2 mm above the nucleus accumbens (from skull surface: anteroposterior 1.8, mediolateral ±0.8, dorsoventral −5.5) (Paxinos and Watson, 1998), and guide cannulae were permanently fastened to the skull using acrylic cement and jeweler's screws. To prevent blockage, obturators were inserted into the guide cannulae and removed before injections. For intra-accumbal microinfusions of aCSF or JQ1, injection cannulae (28 gauge) were lowered through the guide, and infusions occurred over 1 min using a syringe and pump (10 μm, 0.3 μl per side). Injection cannulae were kept in place for 1 min after infusion to limit backflow.

Histological verification of cannula sites

To confirm cannula placements and minimal tissue damage for rats receiving intra-NAc infusions, pontamine sky blue (2% in 0.5 m sodium acetate, 300 nl) was injected through the cannulae before tissue collection, and sections were mounted directly on slides and counterstained with cresyl violet to localize cannula placements and to confirm the absence of gliosis or tissue damage.

Conditioned place preference (CPP)/conditioned place aversion (CPA)

In the CPP experiments, we used similar methods previously used (Sartor and Aston-Jones, 2012). Briefly, the CPP apparatus consisted of two distinct compartments that were separated by a removable divider. In a pretest acclimation session, mice and rats were allowed to freely explore both compartments for 15 min via an opening in the partition. The time spent on each side of the CPP chamber was recorded via EthoVision tracking software. Groups were organized such that mean baseline pretest scores were not different between treatments. Mice or rats that spent >65% of the time on one side of the chamber during the pretest were excluded. Next, mice and rats were conditioned for 3 d. During conditioning, animals were injected with cocaine (10 mg/kg for rats and 15 mg/kg for mice, i.p.) and restricted to one side of the chamber by a solid divider for 30 min, or injected with saline and confined to the other side of the chamber for 30 min. Injections were administered on both sides of the apparatus for each animal in a balanced fashion in morning and afternoon sessions (at least 4 h apart). Vehicle, JQ1, or iBET-151 was injected systemically (10, 25, or 50 mg/kg, i.p.) or intracranially (JQ1, 10 μm) 5 min before each cocaine conditioning session. Intracranial injections of JQ1 outside but near the NAc were used as anatomical controls (bilateral or unilateral misses). To test rewarding or aversive properties of JQ1, a different group of mice were conditioned with JQ1 (50 mg/kg) on one side of the chamber and vehicle on the other side (JQ1/Veh group) or vehicle on both sides of the chamber (Veh/Veh group) for 3 d and were tested for CPP, as described above. After conditioning, mice and rats were given a 15 min (drug-free) preference test.

To determine whether JQ1 affects other types of contextual learning, JQ1 was administered during the acquisition of lithium chloride-induced CPA. First, mice received a 15 min pretest. Next, mice were conditioned for 3 d in a counterbalanced fashion, as described above. During conditioning, JQ1 (50 mg/kg, i.p.) or vehicle was administered 5 min before lithium chloride injection (125 mg/kg, i.p.), and mice were then confined in one side of the CPP chamber for 30 min. The other side of the chamber was paired with a saline injection. The next day, mice were given a 15 min preference test (drug-free). CPP/CPA scores were calculated by measuring the time spent in the drug conditioned side (cocaine, LiCl, or JQ1) during the post-test minus the time spent in the same side during the pretest.

Locomotor activity

Distance traveled was measured in a 27 × 27 cm open field chamber using EthoVision tracking software. Baseline locomotor behavior (distance traveled) was measured in a 30 min habituated test. Mice were grouped such that baseline locomotor activity did not differ between groups. Next, mice were injected with JQ1 (25 mg/kg) or vehicle and placed in the chamber, and distance traveled was measured for 30 min after injection.

Cocaine and sucrose self-administration

Rats were trained daily (2 h/d) to press the active lever in an operant chamber for intravenous delivery of cocaine (0.2 mg/infusion, paired with light cue) under fixed-ratio 1 schedule of reinforcement (FR1 with 30 s timeout after an infusion). Responding at the inactive lever had no scheduled consequences, but inactive presses were recorded. After acquiring self-administration behavior criteria (2 consecutive days with at least 12 infusions earned per day), rats received 10 additional days of cocaine self-administration. For sucrose self-administration studies, rats were trained to press the active lever for 45 mg sucrose pellets (Bio-Serv) under the FR1 schedule of reinforcement, as in cocaine self-administration studies described above. Rats were killed, and brain tissue was collected 24 h after the last session. Control rats were handled daily but did not receive self-administration training.

Western blot

Mice and rats were rapidly decapitated, and the NAc, dorsal striatum and prefrontal cortex (PFC) were dissected over ice and then flash frozen in liquid nitrogen. Samples were homogenized in M-PER lysis buffer (Pierce) with HALT proteinase inhibitors (Pierce). Protein concentrations were determined using a bicinchoninic acid assay kit (Pierce), and 20–30 μg of protein was loaded into precast Tris-HCl polyacrylamide gels (Bio-Rad) for electrophoresis. Protein samples were then transferred to a nitrocellulose membrane and blocked for 30 min in 5% blocking milk (Bio-Rad). Membranes were then incubated overnight at 4°C in blocking milk with the following primary antibodies at a 1:1000 dilution: anti-BRD2 (Cell Signaling Technology, #5848, lot 2), anti-BRD3 (Pierce, #30263, lot PE1859052D), and anti-BRD4 (Santa Cruz Biotechnology, #sc-48772, lot J1310). After multiple washes in 1× TBS, membranes were incubated in their respective HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:1000 to 1:2000) in 5% blocking milk. All protein samples were normalized to GAPDH (Cell Signaling Technology, #2118S, 1:1000). The membranes were imaged with the ProteinSimple Flurochem, and densitometry of protein bands at the appropriate molecular weight was measured using ImageJ.

BDNF ELISA

Approximately 10 million N2a cells were treated with DMSO (vehicle) or JQ1 (100 nm or 1 μm, 48 h) in a 75 mm2 flask. Cells were collected, pelleted, and lysed with M-PER supplemented with HALT proteinase inhibitors. BDNF proteins were measured with the ELISA procedure using the BDNF Emax ImmunoAssay System kit (Promega) according to the manufacturer's instructions. BDNF was normalized to the total protein concentration of each sample using bicinchoninic acid assay kit (Pierce).

Data analysis

GraphPad Prism software was used for graph preparation and statistical analysis. CPP/CPA scores were analyzed by calculating the time spent in the drug-paired side (cocaine, LiCl, or JQ1) on post-test minus the time spent in the same side during pretest. Mean values from CPP/CPA scores, densitometric data from Western blots, and Rq values from qPCR experiments (normalized to controls) were compared between groups using Student's t test or ANOVA. When a significant F value was obtained, comparisons were performed using post hoc analysis. Data are mean ± SEM, and the level of significance was set to 0.05.

Results

To determine whether cocaine regulates BET protein expression, rats received 10 d of cocaine or sucrose self-administration (SA). The average number of active lever presses, inactive lever presses, infusions, and pellets per session are shown in Figure 1, A and B. Using BRD proteins (BRD2, BRD3, and BRD4) as the within-subjects factor and group (naive, sucrose SA, and cocaine SA) as the between-subjects factors, our analysis showed a significant main effect for BRD (F(2,49) = 3.2, p < 0.05), but no main effect for group or interaction between these factors. Bonferroni post hoc analysis revealed a significant difference in BRD4 expression (p < 0.05 for control vs cocaine SA and sucrose SA vs cocaine SA, n = 6–8) (Fig. 1C). No significant difference in BRD2 and BRD3 protein expression was observed between groups.

Cocaine-induced changes in BRD4 binding in the NAc. A, Following repeated cocaine injections, BRD4 binding to the Bdnf promoter was significantly elevated in the NAc, whereas no increase in binding of BRD4 was observed in the promoter regions of Il-1b and Tnf-a (n = 6–8). B, No significant enrichment of BRD3 was observed at the promoters of Bdnf, Il-1b, or Tnf-a following repeated cocaine treatment (n = 6–8). *p < 0.05, significant difference from saline (Bonferroni post hoc test). Data are mean ± SEM.

Discussion

A novel role for BET proteins in cocaine reward

This study reveals a novel role for epigenetic reader proteins in cocaine-mediated behavioral and molecular adaptations. First, we found that the expression of BRD4, but not BRD2 or BRD3, protein was significantly increased in the NAc of mice and rats following repeated, but not acute, cocaine exposure and in the NAc of rats following cocaine self-administration. Although BRD4 expression was only increased 4–24 h after the last drug exposure, the downstream effects of BRD4 may have long-term consequences on unique gene programs that contribute to the addictive state. Furthermore, BRD4 may play a role in multiple psychostimulant-induced neuroadaptations, as one previous study revealed that Brd4 mRNA is upregulated in mesotelencephalic neurons following repeated nicotine injections, although no follow-up experiments were performed (Saito et al., 2005). Second, we revealed that pharmacological inhibition of BET proteins with JQ1 attenuated cocaine CPP in a dose-dependent manner. However, iBET-151, a similar BET inhibitor that does not cross the blood–brain barrier, did not significantly affect cocaine CPP, indicating that the results obtained were due to the effects of JQ1 within the CNS. Third, we confirmed that BET activity in the NAc is essential for cocaine CPP, as injections of JQ1 directly into this region blocked cocaine CPP, whereas injections nearby but outside of the NAc had no effect. Additional behavioral studies revealed that JQ1 did not alter CPA, indicating that not all types of contextual learning behaviors are reduced by JQ1. Administration of JQ1 alone did not induce conditioned aversion or preference to the treatment-associated chamber, nor did JQ1 alter locomotor activity at doses found to affect cocaine CPP. Exploring the underlying mechanisms, we found that JQ1 blocked cocaine-induced elevation in Bdnf in the mouse NAc; and in siRNA studies, we showed that BRD4, but not BRD2 or BRD3, knockdown attenuated the expression of Bdnf in vitro. Further investigation, using ChIP followed by qPCR, revealed that BRD4, but not BRD3, is recruited to promoter region of Bdnf in the mouse NAc following repeated cocaine injections. Together, these findings establish BET proteins as key mediators in the epigenetic response to cocaine and offer new therapeutic avenues for the treatment of cocaine addiction.

The dynamic change in Bdnf expression following cocaine use is accompanied by corresponding epigenetic modifications to the Bdnf gene in specific brain regions. For example, repressive marks and enzymes (H3K9me2, G9a, and MeCP2) are decreased at the Bdnf promoter in the NAc and/or mPFC following cocaine use (Maze et al., 2010; Sadri-Vakili et al., 2010). Conversely, epigenetic marks associated with active transcription, such as H3 acetylation, are increased at the Bdnf promoter in the NAc, mPFC, and VTA following cocaine administration (Kumar et al., 2005; Sadri-Vakili et al., 2010; Schmidt et al., 2012), and in the PFC of male offspring of cocaine-experienced sires (Vassoler et al., 2013). The data presented here add another layer of complexity to the epigenetic regulation of Bdnf in response to repeated cocaine exposure. We show that pharmacological inhibition of BET proteins reduced cocaine-mediated induction of Bdnf expression in the NAc and behavioral response to cocaine, and repeated cocaine injections enhanced BRD4 protein expression and BRD4 binding to the Bdnf promoter in the NAc (the same promoter region that was previously shown to have increased histone acetylation) (Kumar et al., 2005). However, increased BRD4 expression is not necessary for the acute effects of cocaine in the NAc, as inhibition of BRD4 blocked acute cocaine-induction of Bdnf, but acute cocaine did not increase BRD4.

Increasing evidence suggests that various inflammation factors in the brain are also involved in the behavioral effects of abused drugs (Niwa et al., 2007; Cearley et al., 2011), and BET inhibitors are known to attenuate the expression of many of these inflammation-related genes in vitro (Belkina et al., 2013; Meng et al., 2014). Although not statistically significant when comparing all groups, a decreasing trend appears in Il-6 expression following JQ1/cocaine injections versus Veh/cocaine and in Il-1b and Tnf-a following JQ1 versus vehicle injection (Fig. 4). However, we did not observe changes in BRD3 or BRD4 binding to the promoters of these genes in the NAc following repeated cocaine exposure. Timing of tissue collection, cell type-specific mechanisms, secondary effects of JQ1, location of BRD binding (promoter, gene body, etc.), and BRD2-mediated mechanisms (BRD2 ChIP was not performed due to lack of validated antibodies) may explain why we did not observe changes in binding of BRD3 or BRD4 to promoter of these genes, even though JQ1 altered their expression in the mouse NAc. Interestingly, Fosb was increased in the NAc following a single injection of JQ1, which is consistent with previous reports showing that BET inhibition elevates Fosb expression in other cell types (Nicodeme et al., 2010). Because Fosb is associated with cocaine-induced plasticity (Hiroi et al., 1997; Harris et al., 2007), additional studies are needed to identify what regulatory role, if any, BET proteins have in Fosb expression and whether acute or chronic JQ1 exposure leads to alterations in protein levels in the NAc. In addition, genome-wide ChIP analysis of specific BET proteins (e.g., BRD4 ChIP-seq) following repeated cocaine injections and/or self-administration, and behavioral/molecular studies that selectively inhibit individual BET proteins in specific brain regions are needed to better understand the long-term, cocaine-mediated adaptations associated with BET proteins.

BET inhibitors as potential therapeutics for the treatment of addiction

Identifying novel therapeutics for the treatment of addiction is an area of intensive investigation. To date, only a limited number of epigenetic-related targets have been studied in the realm of addiction, and the underlying mechanisms of many of these proteins in cocaine-seeking are unclear. As mentioned previously, HDAC inhibitors have been shown to be effective in multiple addiction-related phenomena, and there has been increasing interest in their use for drug abuse treatment. However, some adverse side effects have been reported in clinical studies using HDAC inhibitors (Subramanian et al., 2010), suggesting that these drugs may not be well tolerated by all patients. In the current studies, we report that BET inhibition attenuates behavioral responses to cocaine but does not affect other types of contextual learning, nor were BET inhibitors rewarding or aversive. Korb et al. (2015) recently showed that chronic JQ1 reduced novel object recognition memory, but not acquisition of fear conditioning, indicating that some additional memories may be affected by chronic BET inhibition. Other preclinical studies, however, have shown that animals can be treated chronically with JQ1 (50 mg/kg a day for 1 month) without major systemic toxicity (Matzuk et al., 2012); and in the multiple ongoing Phase I/II clinical trials, no serious side effects of BET inhibitors have been reported so far (www.Clinicaltrials.gov). Thus, our data, along with other preclinical and clinical data, indicate that BET inhibitors may have therapeutic utility in a number of different pathologies, including addiction, and appear to be generally safe for clinical use, but additional studies are needed to thoroughly investigate potential side effects of chronic BET inhibition.

Footnotes

This work was supported by National Institute on Drug Abuse Grants R01DA035055 and R21DA035592. We thank Andrea Malvezzi, Sandy Vogt, Andrea Johnstone, Chiara Pastori, Marco Magistri, and Sari Izenwasser for assistance. The cocaine used in these studies was obtained from the National Institute on Drug Abuse Drug Supply Program.

The authors declare no competing financial interests.

Correspondence should be addressed to Dr. Claes Wahlestedt,
Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Avenue, BRB-407 (M-860), Miami, FL 33136.CWahlestedt{at}med.miami.edu

(2004) A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal. J Neurosci24:1604–1611, doi:10.1523/JNEUROSCI.5124-03.2004, pmid:14973246.

(2008) The effects of sodium butyrate, an inhibitor of histone deacetylase, on the cocaine- and sucrose-maintained self-administration in rats. Neurosci Lett441:72–76, doi:10.1016/j.neulet.2008.05.010, pmid:18599214.